insights into structural features of plasmodium falciparum...

International Journal of Research on Social and Natural Sciences Vol. I Issue 2 December 2016 ISSN (Online) 2455-5916

1

Journal Homepage: www.katwacollegejournal.com

Insights into structural features of Plasmodium falciparum

4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase

enzyme

Achintya Mohan Goswami, Physiology, Krishnagar Government College, West Bengal, India

Article Record: Received July 16 2016, Revised paper received Nov. 30 2016, Final Acceptance Dec. 4 2016

Available Online December 7 2016

Abstract

Malaria remains one of the most serious infectious diseases in the world. Though there are four species of

Plasmodium genus, but the most responsible and virulent among them is Plasmodium falciparum. The unique

biochemical processes that exist in Plasmodium falciparum provide a useful way to develop novel inhibitors.

One such biochemical pathway is methyl erythritol phosphate (MEP) pathway, required to synthesize

isoprenoids. In the present study a detailed computational analysis has been performed for 4-hydroxy-3-

methylbut-2-en-1-yl diphosphate synthase, a key enzyme in MEP pathway. The structural properties, secondary

structure and evolutionary conservation of the enzyme were studied. The homology model of the enzyme was

also developed.

Key Words: Plasmodium falciparum; 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; Homology

modeling; in silico; Methyl erythritol phosphate pathway

1. Introduction

Malaria is considered as one of the world’s leading causes of morbidity and mortality as evident from 2016 World Health Organization Report, released in December 2015, “there were 214 million cases of malaria in 2015 and 438,000 deaths” (World Health Organization, 2016). Plasmodium

falciparum, a protozoan parasite, is a causative agent of malaria in humans. Malaria caused by this

species (also called malignant or falciparum malaria) is the most dangerous form, with the highest

rates of complications and mortality. As P. falciparum increasingly develops resistances against

commonly used drugs; so identification of novel targets for finding new anti-malarial agents is very

important (Rosenthal & Miller, 2001; Daniel et al., 2012).

P. falciparum, and other members of the apicomplexa phylum, contains an organelle called the

apicoplast. The metabolic pathways in apicoplast differ from the host and therefore apicoplast

metabolic pathways open up new possibilities of anti malarial drug designing. The isoprenoid

metabolic pathway inside the apicoplast is crucial for the P. falciparum survival (Poulter, 2009).

There are two different biosynthetic pathways that have been identified for isopentenyl pyrophosphate

(IPP) and dimethylallyl pyrophosphate (DMAPP) synthesis. One is the well known mevalonate

pathway, which is present in most eukaryotes including mammals, higher plants, and archaea; and the

other is methyl erythritol phosphate (MEP) pathway, which occurs in most bacteria, parasitic protozoa

of the phylum Apicomplexa, plant plastids, and also present in several pathogenic microorganisms

(Rohmer et al., 1993; Eisenreich et al., 2004; Rohmer, 2008; Lombard & Moreira, 2011). The MEP

pathway is active in all intra-erythrocytic stages of the parasite and it is not used by humans (van der

Meer & Hirsch, 2012). Therefore, this unique targetable pathway may be considered for the

development of new drugs against Plasmodium (Jomaa et al., 1999; Wiesner et al., 2008). Enzymes

of the MEP pathway have been thoroughly explored in the last 20 years with respect to their

molecular and functional properties (Grawert et al., 2011). Recent in silico study with Plasmodium

http://www.katwacollegejournal.com/


2

falciparum 1-deoxy-D-xylulose-5-phosphate synthase (an important enzyme in MEP pathway) has

identified ten potential compounds in thiamine diphosphate binding region of the enzyme by virtual

screening of ZINC database (Goswami, 2017). The penultimate enzyme of the MEP pathway is 4-

hydroxy-3-methylbut-2-en-1-yl diphosphate synthase which converts 2-C-Methyl-D-erythritol 2,4-

cyclodiphosphate into (E)-4-hydroxy- 3-methyl-but-2-enyl-diphosphate (Figure 1)

[http://mpmp.huji.ac.il/maps/isoprenoidmetpath.html].

In the present study, Plasmodium falciparum 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase

has been subjected to extensive computational study to determine its chemical and structural features

along with its evolutionary conservation and protein-protein interaction network. The study is also

extended to predict good quality model of the enzyme using homology modeling techniques.

2. Materials and methods

2.1 Sequence retrieval

The amino acid sequence of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase [Accession no.

gi|1016051734|] of P. falciparum 3D7 was retrieved from the National Center for Biotechnology

Information (NCBI). The protein is 824 amino acids long and used for further analysis in the present

study.

2.2 Primary structure details

ExPasy’s ProtParam tool was utilized to calculate the physico-chemical characteristics of 4-hydroxy-

3-methylbut-2-en-1-yl diphosphate synthase (Colovos & Yeates, 1993). Theoretical isoelectric point

(pI), molecular weight, total number of positive and negative residues, extinction coefficient,

instability index (Guruprasad et al., 1990), aliphatic index (Ikai, 1980) and grand average

hydropathicity (GRAVY) of the protein were calculated using the default parameters.


3

2.3 Secondary structure analysis

Secondary structure of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase was predicted by using

the self optimized prediction method with alignment (SOPMA Server) (Guermeur et al., 1999) and

PSIPRED Server (Jones, 1999). Protein’s secondary structural properties include α helix, 310 helix, Pi

helix, Beta Bridge, Extended strand, Bend region, Beta turns, Random coil, Ambiguous states and

other states.

2.4 Evolutionary conservation analysis ConSurf (http://consurf.tau.ac.il/) was used for high-throughput characterization of the functional

regions in the protein (Ashkenazy et al., 2010). The degree of conservation of the amino-acid sites

among 50 homologues with similar sequences was estimated. The conservation grades were then

projected onto the molecular surface of the P. falciparum 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase to reveal the patches with highly conserved residues, often important for

biological functions.

2.5 Network interaction

STRING was used to identify protein-protein interaction partners of 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase (Snel et al., 2000).

2.6 Homology modeling

Homology modeling of P. falciparum 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase was

carried out to predict its three dimensional (3D) structure as the crystal structure of the protein was not

available. So, the 3D structure of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase has been

modeled using homology based modeling. Web based server Swiss Model (swissmodel.expasy.org)

is used for homology modeling of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (Arnold et

al., 2006). To improve the quality of predicted model of 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase, energy minimization was performed with the GROMOS 96 force-field

implementation of DeepView v4.04 tool (Guex & Peitsch, 1997). This force field permits to evaluate

the energy of the modeled structure as well as overhaul distorted geometries through energy

minimization. All computations during energy minimization were done in vacuum, without reaction

field. The predicted 3D structure was visualized by PyMOL (http:// www.pymol.org/). The predicted

model of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase was then validated by PROCHECK

(Laskowski et al., 2001) server. PROCHECK is a popular program used to check the stereochemical

quality of a protein structure. A Phi/Psi Ramachandran plot was obtained from PROCHECK to

validate the backbone structure of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase.

3. Results and discussion

3.1 Primary and secondary structure analysis

A physico-chemical analysis of the P. falciparum 4-hydroxy-3-methylbut-2-en-1-yl diphosphate

synthase protein sequence was done by the Expasy server’s ProtParam tool. The protein had the

theoretical pI of 8.91 with a molecular mass of 95225. The detailed amino acid composition of the

protein is given in Table 1.

Table 1. Details of amino acid composition of P. falciparum 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase

Amino Acid Number of Amino

acids

Percentage

A 25 3.033981

C 14 1.699029

D 42 5.097087

E 65 7.88835

F 33 4.004854

G 43 5.218447

H 15 1.820388

I 80 9.708738


4

K 94 11.40777

L 80 9.708738

M 19 2.305825

N 90 10.92233

P 17 2.063107

Q 17 2.063107

R 30 3.640777

S 39 4.73301

T 35 4.247573

V 48 5.825243

W 3 0.364078

Y 35 4.247573

The protein has a high aliphatic index of 95.66. The theoretical extinction coefficients (at 280 nm

measured in water) of the protein are predicted to be 69525 M-1

cm-1

(assuming all pairs of Cys

residues form cystines) and 68650 M-1

cm-1

(assuming all Cys residues are reduced). The protein has

an instability index of 42.00, which denotes that the protein will not be stable in-vitro because a value

over 40 is considered unstable. The instability index is estimated from a statistical analysis of 12

unstable and 32 stable proteins, where it has been found that occurrence of certain dipeptides are

significantly different among stable and unstable proteins. The predicted grand average of

hydropathicity (GRAVY) of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase was -0.452.

ProtParam computed several parameters analyzing the primary structure of the protein sequence.

These parameters are the deciding factors of the proteins stability and function. Results generated by

secondary structure prediction tool SOPMA shows that the enzyme is dominated by alpha helix ~38

% and ~26 % random coils along with ~24 % extended strands and ~12 % beta turns. Figure 2

showed the secondary structure generated by PSIPRED server.

3.2 Evolutionary conservation analysis

Evolutionary information is of fundamental importance for detecting mutations that affect human

health (Goswami, 2015). ConSurf identifies highly conserved residues, variable residues, functional


5

regions in proteins, taking into account the evolutionary relationships among their sequence

homologues (Ramensky et al., 2002). ConSurf was used for high-throughput characterization of the

functional regions of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase protein. The

colorimetric conservation grades, projected onto the molecular surface of the 4-hydroxy-3-methylbut-

2-en-1-yl diphosphate synthase, revealed the patches with highly conserved residues that were often

important for biological function (Figure 3). The ConSurf analysis also revealed, as expected, that the

functional regions of the protein were highly conserved. It was observed that from residue 109 to 391

and from 710 to 824 were highly conser

3.3 Protein-protein interaction network of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase Protein-protein interaction (PPI) networks have become important for understanding the intricate

molecular mechanisms lying behind the cellular phenomena. The network generation also helps to

design new molecular targets for diseases control.

The protein- protein interacting partners of P. falciparum 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase have been determined by STRING (Figure 4).

During the network prediction, STRING utilizes the reference database of UniProt and predicts

functions of different interacting proteins. PPI network demonstrates that 4-hydroxy-3-methylbut-2-


6

en-1-yl diphosphate synthase interacts with other proteins in a high confidence score; among them are

LytB protein, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-deoxy-D-xylulose 5-

phosphate reductoisomerase (PF14_0641), nucleoside diphosphate kinase (PF13_0349), RNA

binding protein (MAL8P1.101), nucleoside diphosphate kinase putative (PFF0275c), putative 4-

diphosphocytidyl-2c-methyl-D-erythritol kinase (PFE0150c), U3 small nucleolar ribonucleoprotein

(PF14_0042), and putative Prolyl-t-RNA synthase (PFI1240c).

3.4 Homology modeling of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase

The ability of the protein to interact with other molecules or to have different functions depends upon

its tertiary structure. There is no crystal structure of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate

synthase available in the protein data bank. So the 3D structure of 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase has been modeled using homology based modeling. Web based server,

SwissModel Workspace, has been used for homology modeling of 4-hydroxy-3-methylbut-2-en-1-yl

diphosphate synthase. Figure 5 shows the stick (A) and ribbon (B) representation of the protein

structure obtained from the server.

The three dimensional structure is also in agreement with secondary structure implying that the

enzyme is dominated by alpha helix and random coils, followed by beta sheet.

4. Conclusions

Computational study has now got major importance to study protein structure-function relationship at

molecular and atomic level. In this study, in silico analyses have been carried out for P. falciparum 4-

hydroxy-3-methylbut-2-en-1-yl diphosphate synthase to get insights into its structural properties. The

evolutionary conserved regions of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase have been

identified along with the basic structural features. Homology model for 4-hydroxy-3-methylbut-2-en-

1-yl diphosphate synthase has been generated to understand the structure in three dimensional spaces.

Acknowledgement

I want to acknowledge UGC-Minor research project [F. No. PSW-092/14-15 (ERO)] for

infrastructural support.

References

Arnold K., Bordoli L., Kopp J., & Schwede T. (2006). The SWISS-MODEL Workspace: A web-

based environment for protein structure homology modelling. Bioinformatics, 22,195-201


7

Ashkenazy, H., Erez, E., Martz, E., Pupko, T., & Ben-Tal, N. (2010). ConSurf 2010: calculating

evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic

Acids Res, 38, W529–533

Colovos, C., & Yeates, T.O. (1993). Verification of protein structures: patterns of nonbonded atomic

interactions. Protein Sci., 2, 1511–1519.

Daniel, J.P., Amanda, K.L., Daniel, E.N., Stephen, F.S., et al. (2012). Sequence-based association and

selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite. Proc.

Natl. Acad. Sci. U. S. A., 109, 13052–30571.

Eisenreich, W., Bacher, A., Arigoni, D., & Rohdich, F. (2004). Biosynthesis of isoprenoids via the

non-mevalonate pathway. Cell. Mol. Life Sci, 61, 1401–1426.

Gill, S.C. & Von, H.P. (1989). Calculation of protein extinction coefficients from amino acid

sequence data. Anal Biochem, 182, 319–26.

Goswami, A.M. (2015). Structural modeling and in silico analysis of non-synonymous single

nucleotide polymorphisms of human 3b-hydroxysteroid dehydrogenase type 2. Meta Gene, 5, 162–172.

Goswami, A.M. (2017). Computational analysis, structural modeling and ligand binding site

prediction of Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate synthase, Comput. Biol. and

Chem., 66, 1–10.

Grawert, T., Groll, M., Rohdich, F., Bacher, A., & Eisenreich, W. (2011). Biochemistry of the

non-mevalonate isoprenoid pathway. Cell. Mol. Life Sci., 68, 3797–3814.

Guermeur, Y., Geourjon, C., Gallinari, P., & Delage, G. (1999). Improved performance in protein

secondary structure prediction by inhomogeneous score combination, Bioinformatics, 15, 413–21

Guex, N., & Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-PdbViewer: an environment for

comparative protein modeling. Electrophoresis, 18, 2714-2723.

Guruprasad, K., Reddy, B.V., & Pandit, M.W. (1990). Correlation between stability of a protein and

its dipeptide composition, a novel approach for predicting in vivo stability of a protein from its

primary sequence. Protein Eng, 4, 155–61

Ikai, A. (1980). Thermostability and aliphatic index of globular proteins. J Biochem, 88, 1895– 1898.

Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek, B. et al. (1999). Inhibitors of the non-mevalonate

pathway of isoprenoid biosynthesis as antimalarial drugs. Science, 285, 1573–1576.

Jones, D.T. (1999). Protein secondary structure prediction based on position-specific scoring matrices,

J. Mol. Biol., 292, 195-202

Laskowski, R.A., MacArthur, M.W., & Thornton, J.M. (2001). PROCHECK: validation of protein

structure coordinates”, In International Tables of Crystallography, M.G. Rossmann, E.

Lombard, J., & Moreira, D. (2011). Origins and early evolution of the mevalonate pathway of

isoprenoid biosynthesis in the three domains of life. Molecular Biology and Evolution, 28, 87–99.

Malaria Parasite Metabolic Pathways. http://mpmp.huji.ac.il/maps/isoprenoidmetpath.html

Poulter, C. D. (2009). Bioorganic chemistry: a natural reunion of the physical and life sciences.

Journal of Organic Chemistry, 74, 2631–2645.

Ramensky, V., Bork, P., & Sunyaev, S. (2002). Human non-synonymous SNPs: server and survey.

Nucleic Acids Res., 30, 3894–3900.

Rohmer, M. (2008). From molecular fossils of bacterial hopanoids to the formation of isoprene units:

discovery and elucidation of the methylerythritol phosphate pathway. Lipids, 43, 1095–1107

Rohmer, M., Knani, M., Simonin, P., Sutter, B., & Sahm, H. (1993). Isoprenoid biosynthesis in

bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J, 295,

517–524.

Rosenthal, P.J. & Miller, L.H. (2001). The need for new approaches to antimalarial chemotherapy.

Antimalarial Chemother, 82, 3–13.

Snel, B., Lehmann, G., Bork, P., & Huynen, M.A. (2000). STRING, a web-server to retrieve and

display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res, 28, 3442–3444

van der Meer, J.Y., & Hirsch, A.K. (2012). The isoprenoid-precursor dependence of Plasmodium spp,

Nat. Prod. Rep., 29, 721–728


8

World Health Organization (2016). Malaria Fact Sheet Updated April 2016. World Health

Organization, http://www.who.int/mediacentre/factsheets/fs094/en/

Wiesner, J., Reichenberg, A., Heinrich, S., Schlitzer, M., & Jomaa, H. (2008). The plastid-like

organelle of apicomplexan parasites as drug target. Curr. Pharm. Des., 14, 855– 871

insights into structural features of plasmodium falciparum...

Documents